Bats and their ectoparasites (Nycteribiidae and Spinturnicidae) carry diverse novel Bartonella genotypes, China

Abstract Bartonella species are facultative intracellular bacteria and recognized worldwide as emerging zoonotic pathogens. Bartonella were isolated or identified by polymerase chain reaction (PCR) in bats and their ectoparasites worldwide, whereas the association between them was scarce, especially in Asia. In this study, a retrospective analysis with frozen samples was carried out to identify the genetic diversity of Bartonella in bats and their ectoparasites and to investigate the relationships of Bartonella carried by bats and their ectoparasites. Bats and their ectoparasites (bat flies and bat mites) were collected from caves in Hubei Province, Central China, from May 2018 to July 2020. Bartonella were screened by PCR amplification and sequencing of three genes (gltA, rpoB, and ftsZ). Bats, bat flies, and bat mites carried diverse novel Bartonella genotypes with a high prevalence. The sharing of some Bartonella genotypes between bats and bat flies or bat mites indicated a potential role of bat flies and bat mites as vectors of bartonellae, while the higher genetic diversity of Bartonella in bat flies than that in bats might be due to the vertical transmission of this bacterium in bat flies. Therefore, bat flies might also act as reservoirs of Bartonella. In addition, human‐pathogenic B. mayotimonesis was identified in both bats and their ectoparasites, which expanded our knowledge on the geographic distribution of this bacterium and suggested a potential bat origin with bat flies and bat mites playing important roles in the maintenance and transmission of Bartonella.

In the last two decades, a large number of novel viruses have been found in bats (Chen et al., 2014), whereas the study of bacterial agents in bats has been far more neglected (Mühldorfer, 2013).
There was evidence that bat-borne bartonellae were associated with infections in humans and other animals. Bartonella mayotimonensis was first isolated from the aortic valve of a patient with endocarditis in the United States (E. Y. Lin et al., 2010). Subsequently, Bartonella related to B. mayotimonensis were identified in bats from Finland (Veikkolainen et al., 2014), the United States (Lilley et al., 2017), the United Kingdom (Concannon et al., 2005), France, and Spain (Stuckey, Boulouis, et al., 2017). In addition, some Bartonella genotypes found in bats from Georgia were genetically related to those identified in dogs from Thailand and humans from Poland (Urushadze et al., 2017). Moreover, a novel Bartonella species, Bartonella rousetti, was isolated from fruit bats in Nigeria, and serological studies revealed that infection with this bacterium was prevalent in the local population (Bai et al., 2018). These reports highlighted the zoonotic potential of bat-borne bartonellae and underscored the need for expanded surveillance and investigation of these pathogens.
Bats harbour numerous ectoparasites, including bat flies (Diptera: Nycteribiidae and Streblidae), bat bugs (Hemiptera: Cimicidae and Polyctenidae), bat fleas (Siphonaptera: Ischnopsyllidae), bat ticks (Ixodida: Ixodidae and Argasidae), and bat mites (Mesostigmata: Spinturnicidae and Macronyssidae) (Szentiványi et al., 2019). In the last decade, diverse novel Bartonella strains/genotypes were identified in bats and their ectoparasites worldwide ( Figure 1, Table S1). Besides, a recent study inferred that bats had a great influence on both the origin and spread of Bartonella among other mammals and geographic regions (Mckee et al., 2021). However, there was a lack of understanding on the maintenance and transmission of Bartonella in bat populations. Bat flies and bat mites are obligate hematophagous ectoparasites of bats, and they may play important roles in the transmission and maintenance of bat pathogens. Bat flies were the most studied ectoparasites of bats in terms of bartonellae. The presence of identical genotypes of Bartonella in bat flies and their bat hosts was frequently reported (Brook et al., 2015;Dietrich et al., 2016;Judson et al., 2015;Kamani et al., 2014;Qiu et al., 2020); therefore, bat flies were generally considered as vectors for transmitting bartonellae among bats. Few studies reported the occurrence of Bartonella in bat mites (Hornok et al., 2012;Ikeda et al., 2020;Reeves et al., 2016), and a recent study in Poland found that identical genotypes of Bartonella were shared among bats and their Spinturnix myoti mites, indicating the possible role of bat mites in the acquisition and transmission of Bartonella (Szubert-Kruszyńska et al., 2019).
Studies on the genetic diversity of Bartonella in bats and their ectoparasites in Asia were quite limited (Figure 1), and there was a lack of research on the relationship between them. In China, Bartonella were previously identified in bats from Shandong Province by our team (Han et al., 2017) and from Taiwan (J.-W. Lin et al., 2012). However, there were hardly any reports of Bartonella in bat ectoparasites other than F I G U R E 1 Map showing our current understanding of Bartonella in bats and their ectoparasites worldwide. Countries or regions with reports of Bartonella in bats or their ectoparasites are highlighted in gold. The map was created based on data from Table S1 one bat fly in China (Morse et al., 2012). Considering the highly diverse novel Bartonella genotypes identified in bats from China in our previous work (Han et al., 2017), there was a need for further investigation on the relationship of Bartonella in bats and their ectoparasites.
In this study, a retrospective analysis with frozen samples was carried out to identify the genetic diversity of Bartonella in bats and their ectoparasites and to investigate the relationships of Bartonella carried by bats and their ectoparasites. Our study will provide insight into the evolution and ecology of Bartonella in bat populations.

Ethics statement
Collection of bats for microbiological studies was approved by the Ethics Committee of the Medical School, Wuhan University (WHU2020-YF0023), and every effort was made to minimize the discomfort of bats.

Sampling and species identification of bats and their ectoparasites
Frozen bats, bat flies, and bat mites stored in our laboratory, which were sampled for an ongoing programme aiming at identifying pathogens in bats, were used for the analysis of Bartonella. These bats were collected with mist nets, which were settled near the entrance of karst caves at sunset when bats left roosts for night feeding, and bats were collected in the next early morning. Captured bats were put in a bag, sacrificed by inhaling of ethyl ether in the field, and then transported back to the laboratory on ice as soon as possible. Once arrived at the laboratory, bats were checked for ectoparasites with forceps, with bat flies in the fur and bat mites on the membrane wings.
After collection of ectoparasites, thoracic and abdominal organs of bats were collected. All the specimen were stored at −80°C until use. Bat species were preliminary identified by morphology, and then confirmed by polymerase chain reaction (PCR) amplification and sequencing of the cytochrome B (cytB) gene as described previously (Li et al., 2021).

2.3
Molecular detection of Bartonella in bats, bat flies, and bat mites DNA was extracted from bat flies, bat mites, and bat liver tissue samples using QIAamp DNA Mini Kit (Qiagen, Valencia, CA, Spain).
The PCR reaction was performed in a 50 μl mixture containing 0.25 μl 5 U/μl TaKaRa Ex Taq (TaKaRa, Shiga, Japan), 5 μl 10× Ex Taq buffer, 4 μl 25 mM MgCl 2 , 4 μl 2.5 mM dNTP mixture, 5 μl 10 μM each forward and reverse primer (Sangon Biotech, Shanghai, China), 27.75 μl nuclease-free water, and 5 μl sample DNA. PCR was performed with one denaturation cycle at 95˚C for 5 min followed by 40 cycles at 95˚C for 30 s, 55˚C for 30 s, and 72˚C for 90 s, and an additional final cycle at 72˚C for 10 min. Each PCR assay included nucleasefree water as a negative control. PCR products were analyzed by 1.2% agarose gel electrophoresis. Bands of expected size were excised from the gels, and purified using a Gel Extraction Kit (TSINGKE Biological Technology, Wuhan, China). The purified amplicons were cloned into the pMD19-T vector (TaKaRa, Shiga, Japan), and at least three positive clones were selected for sequencing with the universal primers

Phylogenetic analysis
Sequences of interest were imported into MEGA 7.0, and primers were removed after alignment with ClustalW. Phylogenetic trees were constructed based on the neighbour-joining method using the Kimura 2parameter model in MEGA 7.0, and bootstrap values were calculated using 1000 replicates.

Statistical analyses
Since information on the age, sex, reproduction status, or ectoparasite intensity of bats was not collected in this study, statistical analyses were not performed for these factors. Chi-squared test and Fisher's exact test were used to evaluate the Bartonella prevalence by bat species, and differences were statistically significant if p-values <.05.

Sampling and species identification of bats and their ectoparasites
Frozen bat tissue samples and bat ectoparasites used in this study were collected from bats sampled from three cities (Songzi, Jingmen, and Xianning) in Hubei Province, Central China, during May 2018 to July 2020. In each of the three cities, bats were sampled once from a single karst cave (Table 1).
In September 2019, bat flies were collected from 16 bats sampled from a karst cave in Jingzhou, Hubei Province, China. Initially, these TA B L E 1 Sampling information and prevalence of Bartonella in bats, bat flies, and bat mites from Hubei Province, China Note: Asterisk (*) represents pooled samples, and en-dash (-) indicates that no statistical analysis was performed.  Table 1). The 16 bat hosts were identified as Myotis davidii (13) Table 1, and Table S3).

Molecular characterization of Bartonella in bats, bat flies, and bat mites
For pooled samples (bat flies and bat mites) and bats, all the samples were screened for Bartonella with the three genes (gltA, rpoB, and ftsZ).
For individual big bat fly samples, they were firstly screened with the gltA gene. Based on the gltA gene, Bartonella identified in individual big bat flies clustered into only two groups. Therefore, representative samples were further selected for Bartonella characterization by the rpoB and ftsZ genes to reduce workload (Table S3).
Phylogenetic analysis showed that bats, bat flies, and bat mites car-

Statistical analyses
Statistical analyses of Bartonella prevalence by bat species were performed for bats collected from a cave in Xianning in 2020, and the Bartonella prevalence among the three bat species (M. adversus, M. davidii, and R. pusillus) was statistically significant (χ 2 = 8.031, p = .018 < .05).

Nucleotide sequence accession numbers
The

DISCUSSION
Bats, bat flies, and bat mites were collected from Hubei Province in  prevalence of Bartonella in bats in this study was much lower than that reported in previous studies, which may be due to the use of liver tissue rather than blood samples, as Bartonella generally parasitize erythrocytes and endothelial cells (Dehio, 2001), blood samples should be the first choice for PCR screening .  in bats and their ectoparasites in China, our knowledge on the geographic distribution of this bacterium was expanded, and it was very likely that this bacterium was circulating in bat populations with bat flies and bat mites as vectors, even reservoirs. In addition, this study identified a number of novel Bartonella genotypes whose pathogenicity and public health significance were still unknown. It will be necessary to isolate these bat-borne novel bartonellae, evaluate their pathogenicity through animal experiments, and monitor the potential spillover events through the development of detection kits.

Bat flies and bat mites as potential vectors and reservoirs of Bartonella
As described in previous reports (Judson et al., 2015;Sándor et al., 2018;Szubert-Kruszyńska et al., 2019), some Bartonella genotypes were shared by bats and their ectoparasites in this study. Bartonella genotypes identified in bats included I, IV, and VII by gltA, and III, V, XI, and XIII by rpoB, and these genotypes were also detected in bat flies and bat mites. Although the sharing of Bartonella among bats and their ectoparasites could be a result of bloodsucking, more and more evidence suggested that bat flies and bat mites were probably not simple carriers of Bartonella. A recent study showed that the ectoparasites intensity of bats was positively correlated with Bartonella infection, indicating the possible role of these ectoparasites as vectors of Bartonella . In addition, previous studies reported the isolation of Bartonella from bat flies Nabeshima et al., 2020), confirming the viability of Bartonella in bat flies, and highlighting the possibility of bat flies as vectors of Bartonella.
The genetic diversity of bartonellae identified in bat ectoparasites was higher than that in bats in this study, which was also reported in a previous study (Judson et al., 2015). Bat fly/bat mite-unique Bartonella genotypes of this study clustered with bartonellae found in bats in previous studies, it was unlikely that they were symbiotic bacteria of bat flies and bat mites. The absence of some Bartonella genotypes in bats might be partially explained by the low detection rate of Bartonella in bats in this study due to the use of liver tissue rather than blood sam- Although almost all Bartonella genotypes identified in bats in this study were found in bat flies and bat mites, there were still some Bartonella genotypes unique to bats (Figure 7). The role of other ectoparasites such as hard ticks, soft ticks, fleas, and bugs in the transmission of Bartonella in the bat population should be investigated. Interestingly, although bartonellae are generally considered as vector-borne bacteria, they were also identified in the saliva and faeces of bats, indicating that biting and faecal exposure might also contribute to the transmission and maintenance of bartonellae among bat population (Becker et al., 2018;Veikkolainen et al., 2014). Future studies on the infection dynamics of bartonellae will shed light on how these bacteria circulated among bats.

Novel bat fly and bat mite species
Bat flies are obligate bloodsucking ectoparasites that commonly parasitize in the fur and wing membranes of bats. Bat flies are divided into two families: the wingless, spider-like Nycteribiidae, and the more traditionally fly-like Streblidae (Dick & Patterson, 2006). Currently, the family Nycteribiidae consists of 275 species in 21 genera, and the family Streblidae consists of 227 species in 31 genera. The family Nycteribiidae has a worldwide distribution, while the family Streblidae is mainly found in Western Hemisphere (Szentiványi et al., 2019). Based on the COI and 16S rRNA genes, three bat fly species belonging to three genera (Nycteribia, Penicillidia, and Phthiridium) in the family Nycteribiidae were identified in this study, with Phthiridium sp. as a potentially novel bat fly species.
Mites of the family Spinturnicidae are highly specialized bat ectoparasites. The taxonomy of Spinturnicidae was mainly based on morphology, and molecular identification of this family was explored by Bruyndonckx et al. (2009), making species identification of this family possible even for non-experts in morphology. The family Spinturnicidae includes 12 genera. The genus Spinturnix is the most diverse of this family and includes more than 50 named species, most of which are associated with the Old World bats (Orlova et al., 2020). Currently, only two species are recognized in the genus Eyndhovenia, E. euryalis, and E. brachypus, with the latter only morphologically described from Rhinolophus rouxi bats in China (Luong et al., 2021, Yu-Mei Sun, 1986. Eight bat mite species of three genera (Spinturnix, Eyndhovenia, and Paraperiglischrus) in the family Spinturnicidae were morphologically described in China, with four species in the genus Spinturnix (Spinturnix psi, S. myoti, S. sinicus, and S. kolenatoides) (Rui-Yu Ye, 1996;Tian, 2009;Yu-Mei Sun, 1986). Bat mites of this study were molecularly identified as two species in the family Spinturnicidae, with one species belonging to the genus Spinturnix, and the other to the genus Eyndhovenia. Whether the Spinturnix sp. identified in this study is S. sinicus or S. kolenatoides, or just another novel species, and whether the Eyndhovenia sp. identified in this study is the E. brachypus or another novel species need further study. The combination of morphological characteristics and molecular data will get these bat mites better defined in the future.
Due to the lack of knowledge on the morphology of bat flies and bat mites at the beginning of the study, they were not pooled by species for Bartonella analysis, which left a gap in understanding the host-vector specificity of bartonellae. However, differences in the Bartonella genotypes carried by different bat fly species were observed in this study.
Bartonella genotypes (I and VII by gltA; VII and XI by rpoB) were identified in the big bat fly (P. monoceros), while for the small bat fly pools (Nycteribia sp., and Phthiridium sp.), Bartonella genotypes (II, III, VI, and VII by gltA; X and VI by rpoB) were identified (Table S3). Further studies will be needed to reveal the roles of each bat fly or mite species in the transmission and maintenance of Bartonella.